Co‐expression of mutated Jak2 and Calr enhances myeloproliferative phenotype in mice without loss of stem cell fitness
Authors: Christina M. Schueller, Andrea Majoros, Harini Nivarthi, Robert Kralovics
Journal: American Journal of Hematology (2022)

Myeloproliferative neoplasms (MPNs) are a group of acquired hematopoietic stem cell (HSC) disorders driven by mutations that constitutively activate physiologic signal transduction pathways essential for hematopoiesis. Most patients with classical MPNs harbor mutations within the Janus activated kinase 2 (JAK2), calreticulin (CALR), or thrombopoietin receptor (MPL) genes. The occurrence of driver mutations was thought to be mutually exclusive, but double-positive cases have been reported. However, in rare cases where clonal analysis was performed both mutations occurred in independent clones, suggesting mutual exclusivity at the single cell level.1-3 Recently, a single report described one patient who acquired a JAK2 mutation in a CALR mutant clone.4 Although the JAK2 mutation could be detected in HSCs as well as myeloid progenitors, the variant allele frequency was lowest in the HSC population. Although this report suggests that JAK2-V617F and CALR-del52 mutations can occur in a single clone, the effect on HSC fitness remains elusive. In this study, we tested the hypothesis that Jak2-V617F and Calr-del52 mutations are synthetic lethal if they occur in the same HSC. Since synthetic lethality at the HSC level can manifest as loss of competitive fitness over time or cell death, the only means by which such genetic interaction can be detected is using primary genetically engineered HSCs. Therefore, we generated a mouse model in which both mutations are co-expressed in the hematopoietic system, which allowed us to assess the phenotype of such animals and enabled evaluation of HSC fitness in transplantation experiments. We generated conditional knock-in mice that co-express Jak2 and Calr mutations in the presence of Cre recombinase in hematopoietic lineages (vav-iCre) and analyzed their phenotype (Figure 1A). C57BL/6 vav-iCre and Ly5.1/CD45.1 mice were purchased from Charles River Laboratories. Mice carrying Jak2-V617F mutation were previously published.5 and the Calr-del52 transgenic mice were generated in our laboratory.6 The Calr-del52 vav-iCre mice were further bred with Jak2-V617F mutant mice to generate mice expressing both mutations in the hematopoietic compartment. Blood parameters were measured on hematology analyzer scil Vet abc™ (Horiba ABX, Montpellier, France). Single cell suspensions of bone marrow, spleen, and blood were stained, with relevant panels of antibodies (Tables S1–S3), measured at BD LSRFortessa™ (Flow Cytometry - Core Facilities, Medical University of Vienna, Vienna, Austria) and analyzed using FlowJo software (Version 10.7.1; Ashland, Oregon, USA). Double positive offspring were born at expected Mendelian frequency comparable to the single positive littermates, suggesting no signs of synthetic lethality in utero (Figure 1B). The phenotype of the Calr/Jak2 double positive mice was significantly more severe compared to the single mutant mice. Notably, double positive mice showed more pronounced splenomegaly and higher platelet, leukocyte, granulocyte, monocyte, and lymphocyte count in peripheral blood compared to non-mutated or single mutated mice (Figures 1C–G and S1a). Hematocrit, red blood cell count, and hemoglobin were increased compared to non-mutated and Calr mice but did not exceed the values of Jak2 mice (Figures 1H and S1b,c). In line with the thrombocytosis, double mutant mice showed an increased frequency of megakaryocytes in the bone marrow (Figure 1I). Flow cytometry analysis of the blood and spleen revealed an altered cellular composition with a shift toward myeloid lineages (Figures S1d–i and S2a–f). While the stem cell frequency in double mutant mice seemed unaltered, the myeloid progenitor compartment showed a significant expansion of different progenitor types further explaining the excessive production of terminally differentiated myeloid cells (Figures S2g–j and S3a–i). Spleen and bone marrow of double positive mice also showed morphological changes. More obscured follicular architecture and enhanced extramedullary hematopoiesis was detected in the spleen. The bone marrow presented with more prominent megakaryocyte dyspoiesis and altered myeloid to erythroid ratios with no signs of myelofibrosis (Figure S4, reticulin staining not shown). Most importantly, the aggravated myeloproliferative phenotype of double positive mice manifested in lower overall survival compared to the Jak2 and Calr single mutated mice (Figure 1J). Our results are further supported by the report of a double mutant MPN patient who evolved from essential thrombocythemia to advanced myelofibrosis after acquisition of the JAK2-V617F mutation in a CALR-del52 mutated clone.4 To gain further insight into the cellular processes affected by Calr/Jak2 double mutation we compared gene expression of single and double mutated LSK (Lin- Sca-1+ c-Kit+) cells. While double mutated cells showed a very similar gene expression signature as the Jak2 single mutated cells, differential expression in comparison to Calr mutated cells was more pronounced (Figure S5a–d). Gene sets upregulated in double mutated versus Calr single mutated cells, such as coagulation, IL-2/STAT5 signaling, and IL-6/JAK/STAT3 signaling, are consistent with the increased disease phenotype. Although apoptosis is also among the enriched gene sets, the two genes clustering in this set, Btg2, and Ltb are also associated with maintenance of hematopoietic stem cells. Among the downregulated genes, three were associated with mTORC1 and hedgehog signaling. Compared to Jak2 single mutated cells, significantly downregulated genes clustered in heme metabolism and TNF-alpha signaling. Both genes categorized into the pancreas beta cell pathway, Mafb and Pklr are also involved in erythrocyte differentiation and metabolism. High expression of Ebi3 and STAT5 induced genes, that is Socs2 and Cish in the double mutant cells suggest that phenotypic differences are driven by JAK/STAT signaling (Figure S5e). To examine the impact of double mutation on HSC fitness, we performed competitive primary and secondary bone marrow transplantation (Figure S6a). Double positive bone marrow engrafted into recipients equally well as single positive cells suggesting no functional defect at the HSC level. We observed outgrowth of mutant cells in blood, spleen, and bone marrow with most prominent expansion of mutant cells in the myeloid compartment (Figures S6b–c and S7). Transplanted mice also developed an MPN phenotype reminiscent of the one observed in the transgenic animals (Figures S6d–f and S8). To further test if the double positive HSCs maintained their self-renewal capacity over time, we performed secondary transplantation. Double mutant HSCs efficiently engrafted in secondary recipients and mice developed peripheral blood changes indicative of myeloproliferative disease, further suggesting the absence of any long-term defect (Figures S6g, S9 and S10). Thus, the presence of both mutations in HSCs is unlikely to cause synthetic loss of HSC fitness. To date, with a single case being reported, there is no sizable JAK2/CALR double mutated patient cohort available to evaluate the disease phenotype and estimate survival. Our mouse model allowed phenotypic assessment of JAK2/CALR double mutated MPN. We observed a severely aggravated phenotype in double mutated compared to single mutated animals with increased hematocrit similar to Jak2 mutated mice and aggravated megakaryopoiesis and myelopoiesis compared to all other groups. Likewise, the reported patient also progressed to myelofibrosis after acquisition of the second mutation. Therefore, we conclude that the co-occurrence of both mutations might be a negative predictive indicator.4 HSCs did not show any signs of reduced fitness, ruling out mutual exclusivity due to synthetic lethality. Transplantation experiments using Calr-del52 or Jak2-V617F single mutated knock-in mice have been performed in previous studies and are in line with our results. While Jak2 mutant cells were reported to outgrow wt competitors, heterozygous Calr mutated HSCs did not show a significant proliferative advantage compared to wt cells.6-8 In this study we observed a steady increase in myeloid peripheral blood chimerism in all groups with little expansion in the Calr group and strongest expansion in mice that received double mutant bone marrow. The results were similar on the HSC level. In the Jak2 and double mutated group two mice each reached full mutant HSC chimerism in the secondary transplant. In short, double mutant HSCs engrafted equally well or better than their single mutant equivalents. Therefore, we can rule out reduced stem cell fitness as a reason for rare occurrence of double mutant patients. We hypothesize that patients carrying both mutations in one clone might be underdiagnosed as the most common diagnostic routine is single gene analysis for driver mutations in JAK2, CALR, and MPL at diagnosis. In the reported case, a JAK2-V617F mutation was acquired in a CALRdel52 mutated clone 18 years post initial diagnosis.4 This shows that a second driver mutation can be acquired decades later, which might be another factor leading to such patients being undetected since driver mutations are rarely re-analyzed after initial molecular diagnostics. In addition, establishing if the two mutations are monoclonal or bi-clonal, requires an in vitro colony assay. Presence of colonies with both mutations is the formal proof of monoclonal acquisition of the two mutations. However, this procedure is very time consuming and therefore rarely performed. Andrea Majoros and Robert Kralovics designed the study; Christina M Schueller, Andrea Majoros, and Harini Nivarthi performed experiments and collected the data; Christina M Schueller and Andrea Majoros analyzed the data; Christina M Schueller, Andrea Majoros, and Robert Kralovics wrote the manuscript. This study was supported by the Austrian Science Fund, FWF P34451-B granted to Robert Kralovics and P30041-B26 granted to Harini Nivarthi as well as the Austrian Academy of Sciences, DOC fellowship 26008 granted to Christina M Schueller. We thank Jakob Weinzierl and Elisabeth Fuchs for their technical assistance. We also thank Agnieszka Piszczek, Tamara Engelmaier, Julia Klughofer, and Mihaela Zeba for tissue processing and histochemical staining and Anoop Kavirayani for histopathological assessment. Moreover, we thank the CeMM Biomedical Sequencing Facility and Michael Schuster for performing RNA sequencing and data processing. This study was supported by the Austrian Science Fund, FWF P34451-B granted to Robert Kralovics and P30041-B26 granted to Harini Nivarthi as well as the Austrian Academy of Sciences, DOC fellowship 26008 granted to Christina M Schueller. The authors declare that there is no conflict of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. Table S1. List of antibodies used in flow cytometry staining. Table S2. Flow cytometry staining panels for end point analysis of transgenic mice. Single cell suspensions from spleen and bone marrow were stained with two and three different panels, respectively. Table S3. Flow cytometry staining panels for endpoint analysis of bone marrow transplantation experiments. Single cell suspensions from spleen and bone marrow were stained with two and three different panels, respectively. Figure S1. Calr/Jak2 double mutated mice have altered blood composition. The blood composition of 8- to 9-week-old mice of different genotypes, wt vav-iCre (wt), Calr-del52fl/+ vav-iCre (Calr), Jak2-V617Ffl/+ vav-iCre (Jak2) and Jak2-V617Ffl/+/Calr-del52fl/+ vav-iCre (Calr/Jak2) was compared. (a)–(c) Lymphocyte (a), erythrocyte count (b), and hemoglobin (c) were quantified using an automated blood counter. (d)–(i) Percentage of CD19+ B cells (d), CD4+ T cells (e), CD8+ T cells (f), GR-1+ granulocytes (g), CD11b + myeloid cells (h) and CD49b + NK (i) cells among all viable cells. The dots represent values for individual animals; Lines represent mean values for the group and the error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001); the p-values were calculated using ordinary one-way ANOVA test. The gray area between dotted lines represents physiological levels for wt C57BL/6 mice. Figure S2. Calr/Jak2 double mutated mice have altered spleen composition. Cellular composition of spleens from 8- to 9-week-old mice of different genotypes, wt vav-iCre (wt), Calr-del52fl/+ vav-iCre (Calr), Jak2-V617Ffl/+ vav-iCre (Jak2) and Jak2-V617Ffl/+/Calr-del52fl/+ vav-iCre (Calr/Jak2) was analyzed by flow cytometry. Percentage of CD19+ B cells (a), CD4+ T cells (b), CD8+ T cells (c), GR-1+ granulocytes (d), CD11b + myeloid cells (e) and CD49b + NK cells (f), Lin- Sca-1+ c-Kit+ LSK cells (g), Lin- Sca-1+ c-Kit+ CD48- CD150+ long term hematopoietic stem cells (LT-HSC, h), Lin- Sca-1+ c-Kit+ CD48- CD150- short term hematopoietic stem cells (ST-HSC, i) and Lin- Sca-1+ c-Kit+ CD48+ CD150- multipotent progenitors (MPP, j) among all viable cells. The dots represent values for individual animals; Lines represent mean values for the group and the error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001); the p-values were calculated using ordinary one-way ANOVA test. Figure S3. Calr/Jak2 double mutated mice have increased frequency of multipotent and myeloid progenitors in the bone marrow. Cellular composition of bone marrow from 8- to 9-week-old mice of different genotypes, wt vav-iCre (wt), Calr-del52fl/+ vav-iCre (Calr), Jak2-V617Ffl/+ vav-iCre (Jak2) and Jak2-V617Ffl/+/Calr-del52fl/+ vav-iCre (Calr/Jak2) was analyzed by flow cytometry. (a)–(d) Quantification of stem cells and multipotent progenitors among all viable cells: Lin- Sca-1+ c-Kit+ LSK cells (a), Lin- Sca-1+ c-Kit+ CD48- CD150+ long term hematopoietic stem cells (LT-HSC, b), Lin- Sca-1+ c-Kit+ CD48- CD150- short term hematopoietic stem cells (ST-HSC, c), Lin- Sca-1+ c-Kit+ CD48+ CD150- multipotent progenitors (MPP, d). (e)–(g) Quantification of myeloid progenitors (Lin- Sca-1 low c-Kit+) and further stratification into FcRg+ CD34+ common myeloid progenitors (CMP, f), FcRg+ CD34+ granulocyte- monocyte progenitors (GMP, g) and FcRg- CD34- megakaryocyte-erythroid progenitors (MEP, h). (i) Percentage of CD71+ Ter-119 intermediate erythroid progenitors (ProE) among all viable cells. The dots represent values for individual animals; Lines represent mean values for the group and the error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001); the p-values were calculated using ordinary one-way ANOVA test. Figure S4. Calr/Jak2 mice have altered bone marrow and spleen morphology. Sternum (a) and spleen (b) from 8- to 9-week-old mice of different genotypes, wt vav-iCre (wt), Calr-del52fl/+ vav-iCre (Calr), Jak2-V617Ffl/+ vav-iCre (Jak2) and Jak2-V617Ffl/+/Calr-del52fl/+ vav-iCre (Calr/Jak2). After harvesting, organs were placed in Roti®Histofix 4% (Roth) over night at room temperature and moved to 1% Roti®Histofix in PBS for further storage at 4°C. Tissue processing, H&E staining, and pathological assessment were performed by Histopathology department at Vienna Biocenter Core Facilities. Figure S5. Double mutant cells show upregulation in the JAK/STAT signaling pathway. We analyzed the gene expression signature of sorted LSK (Lin- Sca-1+ c-Kit+) cells of the different genotypes. Differentially expressed genes with Log2 fold change ≥1.5 or ≤−1.5 and adjusted p-value ≤0.05 were considered as relevant. (a)–(d) Double mutant cells were compared to the single mutants. Gene set enrichment analysis was performed using Enrichr and genes were compared to the MSigDB Hallmark 2020 database. Individual genes falling in the different gene sets were highlighted in the corresponding color of their bar. (e) Expression of representative genes for the IL-2/STAT5 and IL-6/JAK/STAT3 pathways. Bars represent mean values of two sequencing reactions from one mouse and the error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001); the p-values were calculated using ordinary one-way ANOVA test. Figure S6. Double mutant stem cells retain their differentiation potential in primary and secondary bone marrow transplants. (a) Schematic representation of primary and secondary transplantation experiments. (b) Peripheral blood chimerism in myeloid cells was determined by FACS analysis every 4–5 weeks. (c) Terminal analysis of the bone marrow chimerism by FACS 25 weeks post first transplant. Due to lower survival only two and three of originally five mice could be included in this analysis for Jak2 and Calr/Jak2 groups, respectively. LSK – Lin- Sca-1+ c-Kit-; LT-HSC – long term hematopoietic stem cells (Lin- Sca-1+ c-Kit- CD150+ CD48-); MPP – multipotent progenitors (Lin- Sca-1+ c-Kit- CD150- CD48+), MK – megakaryocytes (c-Kit- CD41+). (d)–(f) Mice which received the primary competitive bone marrow transplant were observed for phenotypic changes in peripheral blood using an automated blood counter every 4–5 weeks. (g) FACS quantification of LT-HSC chimerism of donor mice (primary transplant, 1st) and recipient mice 13 weeks post secondary transplant (2nd). LT-HSC – long term hematopoietic stem cells (Lin- Sca-1+ c-Kit- CD150+ CD48-). The dots represent values for individual animals; Lines represent mean values for the group and the error bars represent standard error of the mean (SEM). The gray area between dotted lines represents physiological levels for wt C57BL/6 mice. Figure S7. Calr/Jak2 cells are dominant in the myeloid compartment in blood and spleen and provide the majority of myeloid progenitors in the bone marrow. FACS analysis of chimerism in blood, spleen, and bone marrow at the endpoint of the primary transplantation experiment (25 weeks). (a) Chimerism in peripheral blood cells was determined: CD19+ B cells, GR-1+ granulocytes, CD11b + myeloid cells, CD49b + NK cells and CD3+ T cells. (b) Chimerism in splenocytes was determined: CD19+ B cells, GR-1+ granulocytes, CD11b + myeloid cells, CD41+ megakaryocytes (MK), and CD3+ T cells. (c) Chimerism in splenic hematopoietic stem and progenitor populations was determined: Lin- Sca-1+ c-Kit+ LSK cells, Lin- Sca-1+ c-Kit+ CD48- CD150+ long term hematopoietic stem cells (LT-HSC), Lin- Sca-1+ c-Kit+ CD48- CD150- short term hematopoietic stem cells (ST-HSC), Lin- Sca-1+ c-Kit+ CD48+ CD150- multipotent progenitors (MPP). (d) Chimerism in short term hematopoietic stem cells (ST-HSC; Lin- Sca-1+ c-Kit+ CD48- CD150) and myeloid progenitor subpopulations (FcRg+ CD34+ common myeloid progenitors (CMP), FcRg- CD34- megakaryocyte-erythroid progenitors (MEP) and FcRg+ CD34+ granulocyte-monocyte progenitors (GMP)) in the bone marrow was determined. The dots represent values for individual animals; Lines represent mean values for the group and the error bars represent standard error of the mean (SEM). Figure S8. Transplanted mice developed an MPN phenotype with peripheral blood changes similar to the transgenic animals. (a)–(h) Blood parameters from 24-week-old mice (the latest time point with 4 mice/group alive) from the primary bone marrow transplantation experiment were analyzed using an automated blood counter. (i) Comparison of spleen size (spleen/body weight) at the endpoint of the primary transplantation experiment (25 weeks). The dots represent values for individual animals; Lines represent mean values for the group and the error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001); the p-values were calculated using ordinary one-way ANOVA test. The gray area between dotted lines represents physiological levels for wt C57BL/6 mice. Figure S9. Calr/Jak2 cells showed persistence in blood, spleen, and bone marrow after secondary transplantation. FACS analysis of chimerism in blood, spleen, and bone marrow at the at the endpoint of the secondary transplantation experiment (13 weeks). (a) Chimerism in peripheral blood: CD19+ B cells, GR-1+ granulocytes, CD11b + myeloid cells, CD49b + NK cells and CD3+ T cells. (b) Chimerism in splenocytes: CD19+ B cells, GR-1+ granulocytes, CD11b + myeloid cells, CD41+ megakaryocytes (MK) and CD3+ T cells. (c) Chimerism in splenic hematopoietic stem and progenitor populations: Lin- Sca-1+ c-Kit+ LSK cells, Lin- Sca-1+ c-Kit+ CD48- CD150+ long term hematopoietic stem cells (LT-HSC), Lin- Sca-1+ c-Kit+ CD48- CD150- short term hematopoietic stem cells (ST-HSC), Lin- Sca-1+ c-Kit+ CD48+ CD150- multipotent progenitors (MPP). (d) Chimerism in different bone marrow populations: LSK – Lin- Sca-1+ c-Kit-; LT-HSC – long term hematopoietic stem cells (Lin- Sca-1+ c-Kit- CD150+ CD48-); ST-HSC – short term hematopoietic stem cells (Lin- Sca-1+ c-Kit+ CD48- CD150), MPP – multipotent progenitors (Lin- Sca-1+ c-Kit- CD150- CD48+), MK – megakaryocytes (c-Kit- CD41+), and myeloid progenitor subpopulations (FcRg+ CD34+ common myeloid progenitors (CMP), FcRg- CD34- megakaryocyte-erythroid progenitors (MEP) and FcRg+ CD34+ granulocyte- monocyte progenitors (GMP)). The dots represent values for individual animals; Lines represent mean values for the group and the error bars represent standard error of the mean (SEM). Figure S10. Mice showed peripheral blood changes indicative of myeloproliferative disease. Terminal analysis of 13-week-old mice which received the secondary transplant. (a)–(h) Blood parameters were analyzed using an automated blood counter. (i) Comparison of spleen size (spleen/body weight). The dots represent values for individual animals; Lines represent mean values for the group and the error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001); the p-values were calculated using ordinary one-way ANOVA test. The gray area between dotted lines represents physiological levels for wt C57BL/6 mice. 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